The thymus has crucial functions in the development of most naive T cells for the peripheral naive T cell pool in humans and rodents. Despite this essential function, the thymus involutes with age, leading to a dramatic reduction in naive T cell output. Thus, aging is associated with increased incidence of potentially life-threatening infections and autoimmune diseases. Our recent findings and the proposed studies indicate that it might be possible to ameliorate such reduced thymopoiesis. To do so, we must first understand the cellular and molecular changes that lead to thymic involution, which is the long-term goal of our research. Clinically, this knowledge will help us develop novel strategies to rejuvenate the thymus and increase thymic output of naive T cells in the elderly or chronically ill, in patients with immunodeficiency diseases such as HIV, and in patients undergoing chemotherapy or bone marrow transplant. The development of T cells or thymocytes in the thymus depends on thymic epithelial stroma and is regulated by transcription factors whose temporal expression is tightly regulated. We hypothesize that Notch signaling is at the apex of the signal cascade that orchestrates the coordinated expression of transcription factors critical for T lineage commitment and differentiation as well as thymic epithelial cell functional maturation. Changes in the temporal expression of transcription factors that control the development of the immature triple negative (TN) thymocytes result in lower thymic naive T cell output. We also hypothesize that thymic epithelial stroma modulates changes in the expression of these transcription factors. The development of TN thymocytes requires T lineage commitment, cell expansion, and rearrangement of T cell receptors beta and alpha. We propose four specific aims: Aim 1, to determine whether T cell commitment is altered in the aged thymus; Aim 2, to determine whether cell expansion is altered during DN thymocyte development; Aim 3, to determine whether rearrangement of T cell receptors beta and alpha are altered in DN development in the aged thymus; Aim 4, to determine how Notch signaling regulates FoxN1 transcription. We have generated transgenic mice for the epithelium-specific transcription factor Foxn1. These mice show absence of age- associated changes in DN development and are a unique tool for delineating the control of expression of transcription factors in aged thymocytes and the function of thymic epithelial stroma in this process. [unreadable] [unreadable] [unreadable] [unreadable]